In vivo studies showed that dendritic cell （DC） dysfunction occurred in tumor microenvironment. As tumors were composed of many kinds of cells, the direct effects of tumor cells on immature DCs （imDCs） are needed ...In vivo studies showed that dendritic cell （DC） dysfunction occurred in tumor microenvironment. As tumors were composed of many kinds of cells, the direct effects of tumor cells on immature DCs （imDCs） are needed for further studies in vitro. In the present study, bone marrow-derived imDCs were incubated with lymphoma, hepatoma and menaloma cells in vitro and surface molecules in imDCs were determined by flow cytometry. Then, imDCs incubated with tumor cells or control imDCs were further pulsed with tumor lysates and then incubated with splenocytes to perform mixed lymphocyte reaction. The DC-dependent tumor antigen-specific T cell proliferation, and IL-12 secretion were determined by flow cytometry, and enzyme-linked immunosorbent assay respectively. Finally, the DC-dependent tumor-associated antigen-specific CTL was determined by enzyme-linked immunospot assay. The results showed that tumor cell-DC incubation down-regulated the surface molecules in imDCs, such as CD80, CD54, CDIIb, CDIIa and MHC class II molecules. The abilities of DC-dependent antigen-specific T cell proliferation and IL-12 secretion were also decreased by tumor cell incubation in vitro. Most importantly, the ability for antigenic-specific CTL priming of DCs was also decreased by incubation with tumor cells. In the present in vitro study demonstrated that the defective abilities of DCs induced by tumor cell co-incubation and the co-incubation system might be useful for future study of tumor-immune cells direct interaction and for drug screen of immune-modulation. Cellular ＆ Molecular Immunology.展开更多
Cytosine deaminase gene of Escherichia coli strain H30 was cloned, and its initiation codon of ’GTG’ was mutated to ’ATG’ by PCR. Prokaryotic recombinant expression vector pBV220CD was constructed. Clone with high...Cytosine deaminase gene of Escherichia coli strain H30 was cloned, and its initiation codon of ’GTG’ was mutated to ’ATG’ by PCR. Prokaryotic recombinant expression vector pBV220CD was constructed. Clone with high enzyme activity were selected by detecting their specific activity of cytosine deaminase. 5FC（5FC, 5fluorocytosine） could induce the lethal toxicity to cells containing active CD gene. DNA sequence analysis indicated that there were 16 altered bases and 5 of them resulted in the alteration of amino acids in predicted peptide by comparing DNA sequence of the clone H30CD11 with high enzyme activity with CD gene reported in Gene Bank.展开更多
文摘In vivo studies showed that dendritic cell （DC） dysfunction occurred in tumor microenvironment. As tumors were composed of many kinds of cells, the direct effects of tumor cells on immature DCs （imDCs） are needed for further studies in vitro. In the present study, bone marrow-derived imDCs were incubated with lymphoma, hepatoma and menaloma cells in vitro and surface molecules in imDCs were determined by flow cytometry. Then, imDCs incubated with tumor cells or control imDCs were further pulsed with tumor lysates and then incubated with splenocytes to perform mixed lymphocyte reaction. The DC-dependent tumor antigen-specific T cell proliferation, and IL-12 secretion were determined by flow cytometry, and enzyme-linked immunosorbent assay respectively. Finally, the DC-dependent tumor-associated antigen-specific CTL was determined by enzyme-linked immunospot assay. The results showed that tumor cell-DC incubation down-regulated the surface molecules in imDCs, such as CD80, CD54, CDIIb, CDIIa and MHC class II molecules. The abilities of DC-dependent antigen-specific T cell proliferation and IL-12 secretion were also decreased by tumor cell incubation in vitro. Most importantly, the ability for antigenic-specific CTL priming of DCs was also decreased by incubation with tumor cells. In the present in vitro study demonstrated that the defective abilities of DCs induced by tumor cell co-incubation and the co-incubation system might be useful for future study of tumor-immune cells direct interaction and for drug screen of immune-modulation. Cellular ＆ Molecular Immunology.
文摘Cytosine deaminase gene of Escherichia coli strain H30 was cloned, and its initiation codon of ’GTG’ was mutated to ’ATG’ by PCR. Prokaryotic recombinant expression vector pBV220CD was constructed. Clone with high enzyme activity were selected by detecting their specific activity of cytosine deaminase. 5FC（5FC, 5fluorocytosine） could induce the lethal toxicity to cells containing active CD gene. DNA sequence analysis indicated that there were 16 altered bases and 5 of them resulted in the alteration of amino acids in predicted peptide by comparing DNA sequence of the clone H30CD11 with high enzyme activity with CD gene reported in Gene Bank.
